In-depth analysis of Klebsiella aerogenes resistome, virulome and plasmidome worldwide

Klebsiella aerogenes is an emergent pathogen associated with outbreaks of carbapenem-resistant strains. To date, studies focusing on K. aerogenes have been small-scale and/or geographically restricted. Here, we analyzed the epidemiology, resistome, virulome, and plasmidome of this species based on 561 genomes, spanning all continents. Furthermore, we sequenced four new strains from Brazil (mostly from the Amazon region). Dozens of STs occur worldwide, but the pandemic clones ST93 and ST4 have prevailed in several countries. Almost all genomes were clinical, however, most of them did not carry ESBL or carbapenemases, instead, they carried chromosomal alterations (omp36, ampD, ampG, ampR) associated with resistance to β-lactams. Integrons were also identified, presenting gene cassettes not yet reported in this species (blaIMP, blaVIM, blaGES). Considering the virulence loci, the yersiniabactin and colibactin operons were found in the ICEKp10 element, which is disseminated in genomes of several STs, as well as an incomplete salmochelin cluster. In contrast, the aerobactin hypervirulence trait was observed only in one ST432 genome. Plasmids were common, mainly from the ColRNAI replicon, with some carrying resistance genes (mcr, blaTEM, blaNDM, blaIMP, blaKPC, blaVIM) and virulence genes (EAST1, senB). Interestingly, 172 genomes of different STs presented putative plasmids containing the colicin gene.


Brazilian K. aerogenes antibiotic susceptibility
The four Brazilian strains of K. aerogenes were tested for various antibiotics, including carbapenems and cephalosporins (Table 2).The Ka-04RR strain showed resistance to a greater number of antibiotics, mainly cephalosporins and β-lactams.The punctual pattern of resistance to carbapenems, cephalosporins and β-lactams suggests the absence of an enzymatic resistance mechanism.Even so, all strains would be considered multidrug resistant (MDR) 19 .
Among the proteins analyzed, amino acid replacements and/or premature translation stops in AmpD, AmpR, Omp35, and Omp36, which could lead to a phenotype of greater resistance to β-lactam, were present in 148 genomes spread across the phylogeny, regardless the ST (Fig. 1).Most of these genomes (117/148; ~ 80%) did not contain ESBL (19 of these 148 genomes had ESBL) and/or carbapenemase (12 of these 148 genomes had carbapenemase) genes, suggesting that these mutations could be selection mechanisms in the absence of enzymatic resistance genes.Even more so because most of these genomes (100/148; ~ 67%) also lacked acquired resistance  www.nature.com/scientificreports/genes (Table S1).Thus, contrasting to the low prevalence of acquired genes, a large proportion of genomes carrying chromosomal alterations associated with resistance to β-lactam was identified.Integrons (n = 139) could be identified in 96 genomes, mainly from ST93 (n = 29) (Table S10), where almost all were class 1 and only one class 3 integrons (strain 24A19CPO031, DAJACA010000050.1).As integrons are known to capture resistance genes, we searched for these genes in the identified integrons (Table S10).Different combinations of gene cassettes related to resistance were present, where most integrons presented the dfrA gene, while a smaller proportion presented qacE, ant, aac, aph, arr, bla OXA , and bla GES , some of them presenting several alleles.Furthermore, bla VIM and bla IMP carbapenemase genes were also present in gene cassettes (Table S10).Based on the gene cassette sequences of the integrons, 97 of them could be grouped into 22 clusters, with several of them present in genomes of different STs (Table S10).

K. aerogenes virulence analysis
Among the virulence factors identified in the genus Klebsiella, the yersiniabactin, colibactin, and aerobactin loci represent clinically important features.Thus, considering the 561 K aerogenes genomes, 222 genomes co-carried the ybt and clb loci, and 16 carried the ybt loci, while only one genome presented the aerobactin loci (strain 2022LN-00016, GCA_023553355.1) (Table S1).Furthermore, among the major STs, these loci were prevalent in the genomes of pandemic clones ST93 (n = 114) and ST4 (n = 47) (Table 3).Almost all ybt and clb loci were in the context of ICEKp10 (208/238 ybt+ genomes), regardless of ST, with the majority of ST4 having the ybt type 17 (n = 45), and majority of ST93 having the ybt type 17 (n = 59) and ybt type 20 (n = 52) (Table S1), while only the clb type 3 was found associated with these loci.ICEKp other than ICEKp10 were identified in a few genomes and carried only the ybt, lacking clb (Table S1).Only one environmental genome (ST93) presented the ybt 17 and clb 3 loci in the context of ICEKp10, while other animal and environmental genomes lacked these virulent loci.Curiously, the salmochelin (iro) virulence trait is present in almost all K. aerogenes genomes (517/561; ~ 92%) (Table S1), of which 88 did not present the entire cluster (iroBCDEN).In addition, although lacking the ybt and clb loci, most animal and environmental genomes carried the iro loci (19/27; ~ 70%).Another prevalent virulence factor among the genomes was the Type VI Secretion System (T6SS), present in 553/561 (~ 98%).Regarding toxins, two factors (EAST1 and senB) were found in the genomes of ST240, ST15, ST93, ST331, ST364.In short, ICEKp10 carrying the ybt and clb is widespread in different STs, prevailing in ST4 and ST93.
In four genomes (GCA_020982565.1,GCA_001631645.1, GCA_003952125.1, GCA_021902315.1), the ybt type 4 was predicted, this type being associated with plasmid origin (Table S1).However, analyzing these genomes, we were unable to identify the plasmid replication genes.This could be due to genome assembly fragmentation, however, the ybt loci of these genomes were close to integrases and flanked by tRNA-Asn (in 3/4 genomes), which is a feature associated with chromosomal insertion.Thus, if ybt type 4 is on a mobile element, it appears to have the ability to integrate chromosome.

K. aerogenes plasmid resistome/virulome
We explored the 561 genomes for potential plasmids based on plasmid replicons, and identity and coverage with previously reported plasmids.Contig sequences presenting plasmid replicons and with high identity and coverage to previously reported complete plasmids were found in 317 genomes (317/561; 56%), which totaled 541 putative plasmid sequences (Table S11).Although it is not possible to state whether these contigs represent entire plasmids or fragments thereof, the presence of the plasmid replicon associated with high identity and coverage to previously reported plasmids suggests that these contigs belong to elements with an extra-chromosomal nature.The putative 541 plasmids had a median size of 10 kb and 52% GC content; ranging from 1 to 6 per genome (median of one plasmid); and ColRNAI was the most common replicon type.Based on the mobility markers present in these putative plasmids, we predicted that 51 would be conjugative (44.9 kb median size, 47% GC content), as they carried relaxase, virD4, and virB4 homologues genes 26 ; 339 would be mobilizable (9.4 kb median size, 55% GC content) due to the presence of oriT sequences and/or relaxase genes, without T4SS-like genes; and 151 would be non-mobilizable (16 kb median size, 49% GC content) (Table S11).These putative plasmids were mainly harbored in the ST93 (n = 93) and ST4 (n = 34) genomes but were also present in other STs in smaller numbers (n < 10).These genomes are from several continents, presenting a wide time scale (2002 to 2021).Interestingly, most of the putative plasmids carrying the ColRNAI replicon (172/194; ~ 88%) had a median length of 9.2 kb and a similar structure, mainly comprising a cloacin gene (a type of colicin) and its immunity gene, traM, mobC, relB/relE toxin/antitoxin, and rop genes (Fig. 2).Variations were observed in these mobilizable sequences, for example, the sequence DAFYDZ010000031.1 presented genes related to mercury resistance; FKIV01000020.1 presented the bla TEM gene; and Tn3 transposase was present in sequence LULD01000059.1 (Fig. 2).Virulence genes were rare in the putative plasmids, where only five had one gene, including the traJ (associated with invasion), EAST1 (exotoxin), and senB (exotoxin) genes (Table S12).

Discussion
There is a historical gap in the epidemiology of K. aerogenes, as it has always been considered an opportunistic pathogen, but recently interest in this organism has increased, mainly due to the emergence of carbapenemresistant isolates 3 .Furthermore, this species was recently renamed, being previously known as Enterobacter aerogenes, which still influences researchers to keep the old species name, even in recent publications.However, in genomic information databases such as GenBank, this has been updated.Until now, studies focused on this emerging pathogen were small-scale, considering few genomes and/or with a restricted geographic perspective.Here, in addition to generating new genomes of this species in Brazil and contextualizing them in the global scenario, we carried out an in-depth analysis of the resistome, virulome, and plasmidome in view of the emergence of K. aerogenes as a pathogen.
Previous genomic epidemiological analyzes have shown that K. aerogenes ST4 and ST93 have been implicated as the dominant global clones 4 , as have occurred in a few countries in the Americas, Europe, and China.In fact, here, expanding this analysis, encompassing all K. aerogenes genomes, these STs continue to represent the dominant clones, causing infections/outbreaks in several other countries (e.g.Belgium, Germany, Japan, Qatar, Singapore, South Korea, Switzerland, Thailand, United Kingdom), thus characterizing them as pandemic clones that impact the clinics.
K. aerogenes genomes can be clustered by the presence/absence of two virulence determinants: the yersiniabactin and colibactin 5 .In fact, it is observed that most STs lack these loci, while they are predominant in the ST4 and ST93 genomes.These virulence loci were found in several ICEKp, mainly in ICEKp10, and their distribution is not homogeneous among all genomes, even within the same ST, which shows their mobile characteristic.In K aerogenes, ICEKp10 had already been identified in a few strains of ST4 and ST93 from the USA 4 .The present analysis revealed a wide distribution of this virulence-associated element within this species, particularly in ST4 and ST93 genomes from several countries around the world.Moreover, colibactin, a genotoxin that can induce DNA damage in eukaryotic cells and tumor formation 27 , raises concerns about its high prevalence and distribution in Klebsiella species.
to the ybt and clb loci, the salmochelin loci (iro), another virulence trait identified, present in a high proportion of genomes (~ 92%), including those of animal and origin.This result is similar to the findings of other studies considering smaller genome sets 5,28 , and suggests that K. aerogenes may be a potential reservoir of virulence genes for other bacteria 28 .In fact, in K. pneumoniae, salmochelin is detected in low prevalence, being more identified in mobile elements, such as plasmids 29 .Although prevalent, 88 K aerogenes genomes had incomplete salmochelin loci.In avian pathogenic Escherichia coli, it was observed that the absence of iroC, iroDE, or iroN abrogated the virulence of the bacteria 30 .Thus, it can be hypothesized that the iro loci in these K. aerogenes would not act on virulence as in E. coli.Furthermore, the presence of incomplete loci may also suggest that other functions could be involved, such as colonization and survival linked to commensalism, as they can increase the fitness of strains within a specific niche.
A putative virulence island, carrying the aerobactin loci (iucA-D and iutA) and rmpA2 gene, was found in a K. aerogenes genome (GCA_023553355.1).This island has also been observed in several K. pneumoniae plasmids.In fact, recently, a transposon harboring the aerobactin operon was identified in K. pneumoniae virulence plasmids 31 .Most of these plasmids harbor the rmpA/rmpA2 genes, aerobactin, and salmochelin loci, which greatly enhance the virulence of K. pneumoniae strains 32 .Although salmochelin is strongly associated with aerobactin in K. pneumoniae 33 , this was not observed in K. aerogenes as iro was prevalent and iuc rare.Thus, we show here that a mobile element carrying K. pneumoniae hypervirulence marker genes is being transferred between species of the genus Klebsiella.
Although most genomes did not carry acquired antibiotic resistance genes, having only the intrinsic resistome, this does not mean an absence of resistance, since in vitro analyses of Ka-04RR showed its MDR profile, and further in silico analyses revealed the presence of alterations in chromosomally encoded factors associated with this MDR profile.Indeed, chromosomal alterations have been observed in several other genomes.Most genomes (74%; 411/561) do not carry carbapenemase genes (including Ka-04RR), however, almost all of them carry the ampC gene, which if overexpressed could lead to carbapenem resistance 3 .In fact, AmpC is chromosomally encoded by K. aerogenes, and alterations in its regulatory genes could affect AmpC translation 21 .Here we observed several amino acid replacements in AmpC regulatory proteins, some of them already associated with resistance to carbapenems (mainly in AmpD), in addition to a large set of other yet to be characterized in further studies.Moreover, ~ 21% of the genomes showed truncated Omp35 and Omp36 porins, which could also increase resistance to β-lactam agents 3,34 .Therefore, regardless of the absence of β-lactamase genes in most genomes, we raised evidence of other possible mechanisms that could confer some level of resistance to β-lactams in K. aerogenes circulating the world.Unlike K. pneumoniae, reports of integrons in K. aerogenes are scarce [35][36][37] .Here, this genetic element was identified in ~ 17% of the analyzed genomes, presenting several combinations of gene cassettes, some not yet reported in this species, such as bla OXA-1 , bla OXA-10 , bla IMP , bla VIM , bla GES .Thus, carbapenemase genes can also be captured by these genetic platforms in this species.
In Brazil, studies on K. aerogenes have focused on molecular analyses of resistance genes from carbapenemresistant isolates.These studies cover isolates from three Brazilian regions (Minas Gerais, Paraná, and Pernambuco states), most of them co-harboring the bla KPC-2 and bla TEM genes [38][39][40] .In fact, we observed that several Brazilian of ST4 and ST93 genomes presented these genes (Table S1), and plasmids carrying carbapenemase genes (bla KPC-2 , bla NDM-1 ) have already been observed among K. aerogenes in the country 41,42 .In addition, we provided new genomes from other regions, expanding regional epidemiology.Although data on resistance genes in K. aerogenes in Brazil are available, most studies have not defined the ST of these strains, with only reports of ST93 and ST16 10,43 .Thus, the current epidemiological scenario of K. aerogenes in Brazil is mainly driven by ST93, ST16 and also by ST4, as is the case worldwide.In addition, a clinical bla NDM -producing K. aerogenes was recently identified in the country belonging to ST128 44 .Here, ST128 was also associated with environmental (Asia) and animal (Africa) strains, therefore revealing the One Health trait of this species, particularly of ST128.Furthermore, this ST is widespread and evolving in the context of the acquisition of resistance and virulence genes.
In China, bla NDM alleles were associated with plasmids in K. aerogenes ST4 45,46 .Here, the bla NDM gene was also found in several putative plasmids from Singaporean genomes.Interestingly, both countries were the only ones to present genomes with putative plasmids carrying the mcr gene (colistin resistance).Indeed, the initial report of mcr in plasmids occurred in China (2016) 47 , and our analysis showed that the presence of the mcr gene in putative plasmids of K. aerogenes still seems to be restricted to Asia.Even though these putative plasmids are already widespread in E. coli worldwide.Although plasmids drive the exchange of antibiotic resistance in other countries, we could only observe five Brazilian ST93 genomes harboring putative plasmids with bla KPC-2 .Therefore, other transfer mechanisms may be acting in other acquired genes.Nevertheless, if plasmids are also driving K. aerogenes adaptation in Brazil, more sequences should be made available to determine this question, in addition to more data for epidemiology.
The most common type of plasmid replicon identified was the ColRNAI, as observed by Passarelli-Araujoa et al. 5 on a smaller set of genomes.Interestingly, 172/194 of ColRNAI replicon putative plasmids have been observed carrying colicin E3 (cloacin-like; rRNase activity) and its immunity gene.Colicins are bacteriocins produced by some gram-negative bacteria, showing antibacterial activity against closely related species, and being highly found in natural populations of E. coli 48,49 .Indeed, blasting cloacin-bearing K. aerogenes putative plasmids, several E. coli and K. pneumoniae plasmids showed high coverage and identity of > 75% and > 99%, respectively.It has been reported that Cloacin-like exhibits a weaker effect and narrower spectrum of activity against several species, but exhibits high inhibitory activity against K. aerogenes, which suggests that these plasmids are associated with the ecology of this species 49 .Interestingly, most K. aerogenes genomes contained the iutA gene, which encodes a receptor for aerobactin, a rare virulence factor in the current dataset.However, curiously, this receptor is also used by cloacins 49 , suggesting that the main function of this receptor in K. aerogenes may not be related to virulence, but perhaps to ecological functions.
In conclusion, K. aerogenes is a species capable of acquiring resistance genes from other organisms, but it seems to be more prone to becoming resistant by mutations in housekeeping genes, something that could only be assessed by in-depth analysis since most of the in silico resistome survey platforms are not up to date regarding this information.Furthermore, there are K. aerogenes lineages circulating around the world carrying virulence genes that may be determinants for its emergence as a clinical pathogen.

Public data set
All reported Klebsiella aerogenes genomes (n = 557), including complete and draft ones, were obtained from the Genome database of the National Center for Biotechnology Information (NCBI) in October 2022.The accession numbers are supplied in Table S1.

Isolates and genome sequencing and assembly
In this study we analyzed four Klebsiella aerogenes strains from nosocomial cases in the Amazonic and Southeast regions of Brazil: Ka-01RR (subclavian vein catheter tip), Ka-02RR (tracheal secretion), Ka-04RR (unknown), Ka-06RJ (sputum).The genomic DNA extraction was done using the NucleoSpin Microbial DNA kit (Macherey-Nagel), and the genomic libraries were constructed using Nextera paired-end library.The sequencing was performed using Illumina Hiseq 2500, generating reads of 150 bp length.The raw reads were filtered and trimmed using NGS QC Toolkit v.2.3.3 50with a Phred score ≥ 20.The genomes were de novo assembled using SPAdes assembler v3.14.1 51 .

Genome characterization
The K. aerogenes genomes were submitted to the Kleborate v2.1.0 33pipeline to sequence typing, and identification of acquired virulence and antibiotic resistance genes.Integrons were survey with Integron Finder 54 .Type VI Secretion System (T6SS) was identified using the T6SS prediction tool 55 .Insertion Sequences and transposases associated with antibiotic resistance genes were identified using TnFinder 56 .

Figure 1 .
Figure1.Maximum-likelihood tree based on the core gene alignment of the 561 K. aerogenes genomes.The ST number of each genome is next to the accession number.There are two orbits of colored blocks, where the innermost represents the source of isolation, and the outermost represents the regions of the genomes.Next, five colored circles indicate genomes with yersiniabactin (green), colibactin (beige), integron (purple), cloacin (blue), nonsense mutations in AmpD, AmpR, Omp35/36 (red)4,20,21 .The genomes sequenced here are in green background color.Red circles at branches indicate bootstrap values > 80%. https://doi.org/10.1038/s41598-024-57245-1

Figure 2 .
Figure 2. Synteny variations of putative K. aerogenes ColRNAI plasmids.A comparison of putative plasmids is shown, where the cloacin gene and its immunity gene, traM, mobC, relB/relE toxin/antitoxin, and rop genes appear to represent a core segment.

Table 1 .
Assembly information of sequenced Brazilian genomes.

Table 3 .
Prevalence of virulence loci in the main STs of K. aerogenes.